DMA replication and repair are critical for maintaining genome stability. These processes are in part dependent on the activities of an emerging family of structure-specific nucleases. These enzymes, typified by flap endonuclease-1 (FEN-1), possess flap-specific endo- and nick-specific exo- nuclease activities as well as a newly identified gap-dependent endo-nuclease activity. It interacts with 19 different DNA metabolic proteins. The enzyme becomes localized in the nucleus in a cell cycle-dependent manner and in response to DNA damage such as UV and ionized radiation. FEN-1 nuclease is required for the removal of RNA primers during lagging- strand DNA synthesis, DNA damage repair and apoptotic DNA fragmentation. Dysfunction of Fen1 gene results in a strong mutator phenotype. It was predicted that the deficiency of the gene in mammalian cells will lead to formation of cancers. The broad and long-term goal of this etiological study is to link the functional deficiency of this critical DNA replication and repair mutator gene to the formation of genetic diseases such as cancers, to clarify the molecular mechanisms of any such pathology, and to provide a solid foundation for the development of new regimens for cancer therapy. For the last funding cycle, we have identified a novel nuclease activity of FEN-1 and defined numerous clusters of amino acid residues, which are important for different biochemical functions. We were able to establish mouse models to demonstrate that the point mutations lead to high cancer incidence. The current competitive newel application is to further test the hypothesis if various functional alterations in FEN-1 nuclease in the form of mutations and polymorphisms, will result in different genetic susceptibilities in the human population to environmental stresses, which subsequently leads to individual differences in the timing of cancer initiation and progression. The major experimental systems including molecular enzymological, yeast and mouse genetic techniques have been established in the Pi's laboratory. The proposed experiments are therefore designed to establish a linkage among multiple biochemical activities, pathways, and mutagenic and carcinogenic mechanisms employing a set of mutations identified in cancer cells as well as prevalent polymorphisms of the Fen1 gene. Three specific aims are designed: i) to determine if mutations identified in cancer cells and polymorphisms alter the biochemical activities, protein-protein interactions in vitro and mutator phenotypes in yeast cells;ii) To determine how post-translational modifications of FEN-1 affect its nuclear and/or nucleolar localization, and to identify the role of sub-cellular localization in the biological functions of FEN-1;and iii) To model multiple Fen1 mutations and polymorphisms in mice to determine their significance for cancer susceptibility, using human Fen1 functionally defective point mutations identified in cancers and prevalent SNPs in the coding region. Completion of this research will not only clarify the molecular mechanisms underlying cancer pathology, but also provide a solid foundation for development of new regimens for cancer therapy.